PROJECT SUMMARY Agonist antibodies, capable of initiating cellular signaling events through interaction at the cell surface, have emerged as a new paradigm in disease treatment with several promising candidates in clinical trials including T cell activating antibodies. However, the discovery of rare antibodies possessing specific biological functions remains a major biotechnological bottleneck. To address this issue, I seek to employ direct function-based antibody screening methods, an emergent biomedical technology using mammalian intracellular reporter systems for which proof-of-concept has recently been shown for the discovery of agonist antibodies. I believe that this innovative technology has the potential to be revolutionary if it can be generalized. Thus, the goal of the proposed research is to establish new fundamental methods for broad use in function-based antibody screening, which could ultimately lay the foundation for advancing disease treatment. I have recently developed novel model systems that enable robust detection of intracellular signaling via the NF-?B pathway initiated from extracellular activation of the T-cell receptors Ox40 and CD137. These receptors were chosen for their availability of well-defined control agonist antibodies that are invaluable for system validation. I propose to use these model systems to gain new understanding of the fundamental mechanisms and principles involved in function-based antibody screening and employ this knowledge toward the development of new biomedical technology and novel therapeutics via three distinct aims. First, I propose to critically evaluate the relationship between biological activity, antibody display level, receptor display level, and antibody molecular format to identify screening methods that ultimately improve the efficiency of agonist antibody discovery. Second, I propose to develop new Ox40 and CD137 reporter cell systems for simplified and improved function-based antibody discovery via genetic engineering of autocatalytic agonist antibody expression that depends on receptor-specific intracellular signaling in order to improve agonist antibody discovery by both simplifying signal detection and amplifying true positive signals. Thirdly, I seek to identify new Ox40 and CD137 agonist antibodies with improved activity by direct function-based screening. Toward this goal, I will employ computational approaches to inform library design in order to increase the likelihood that library variants effectively engage T cell receptors near the active site. I will employ next-generation sequencing to identify lead candidates, which will then be rigorously evaluated via classic T cell activation assays. Finally, I will critically analyze lead sequences in relationship to agonist activity in order to uncover potential molecular determinants of agonist activity and structure-function relationships. Overall, the proposed research has the potential for broad therapeutic impact given that insufficient cellular signaling is involved in a wide range of pathologies including insufficient wound healing, autoimmune disease and cancer.